Received: 21 March 2018 | Revised: 28 August 2018 | Accepted: 11 September 2018

DOI: 10.1111/acel.12853

REVIEW

Age‐dependent changes and biomarkers of aging in

Caenorhabditis elegans

Heehwa G. Son1 | Ozlem Altintas2 | Eun Ji E. Kim1 | Sujeong Kwon1 |

Seung‐Jae V. Lee1,2

1
Department of Life Sciences, Pohang
University of Science and Technology, Abstract
Pohang, South Korea Caenorhabditis elegans is an exceptionally valuable model for aging research because
2
School of Interdisciplinary Bioscience and
of many advantages, including its genetic tractability, short lifespan, and clear age‐
Bioengineering, Pohang University of
Science and Technology, Pohang, South dependent physiological changes. Aged C. elegans display a decline in their anatomi-
Korea
cal and functional features, including tissue integrity, motility, learning and memory,
Correspondence and immunity. Caenorhabditis elegans also exhibit many age‐associated changes in
Seung‐Jae V. Lee, Department of Life
the expression of microRNAs and stress‐responsive genes and in RNA and protein
Sciences, Pohang University of Science and
Technology, Pohang, Gyeongbuk, South quality control systems. Many of these age‐associated changes provide information
Korea.
on the health of the animals and serve as valuable biomarkers for aging research.
Email: seungjaelee@postech.ac.kr
Here, we review the age‐dependent changes in C. elegans and their utility as aging
Present address biomarkers indicative of the physiological status of aging.
Ozlem Altintas, Institute of Intensive Care
Medicine, University Hospital Zurich,
University of Zurich, Zurich, Switzerland.

Funding information
National Research Foundation of Korea,
Grant/Award Number: NRF-
2016R1E1A1A01941152

1 | INTRODUCTION
Living organisms undergo many degenerative and deleterious func-
tional and structural changes with aging. In animals, these changes
include reduced motor activity, overall metabolic rates, and cognitive
function, as well as impaired digestive function, and increased DNA
damage. Delaying or reversing these degenerative changes is critical
because one of the important goals of aging research is to prolong
the healthy longevity of humans. To achieve this goal, a key initial
step is to define and characterize age‐dependent physiological and
molecular changes. However, it takes a very long time for most mam-
mals to display such age‐dependent changes. Therefore, short‐lived
organisms have been used as valuable models for aging research.
Caenorhabditis elegans is one of the outstanding model organ-
isms used for aging research because of its very short lifespan
and simple physiology. Moreover, experiments with C. elegans are
free of ethical concerns. Many breakthrough discoveries in the
field of aging research have been made using C. elegans because
of these advantages (Kenyon, 2010). To date, the measurement of
lifespan is the most popular method used to study aging in C. ele-
gans. However, this assay still takes a relatively long time because
the lifespan of wild‐type C. elegans is approximately 3 weeks. Addi-
tionally, chronological age does not always reflect biological (func-
tional and physiological) age; a cohort with the same chronological
age undergoes biological changes at variable rates, especially in
the later stages of life. Therefore, defining quantitative changes
associated with physiological aging, referred to as biomarkers of
aging, is crucial for expediting the development of potential anti‐
aging therapies.

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© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

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https://doi.org/10.1111/acel.12853
2 of 11 | SON ET AL.

Legitimate biomarkers of aging should exhibit changes in expres-
sion level or activity during physiological aging and therefore serve
as a reference to predict the status of aging without harming the
subjects. Thus, prominent age‐dependent molecular and physiological
changes are reasonable candidates for biomarkers of aging. The char-
acteristics of aging in C. elegans have been described in another
review article (Collins, Huang, Hughes, & Kornfeld, 2008). In addi-
tion, a recently published book chapter is an outstanding resource
for the age‐dependent anatomical changes of C. elegans (Herdon,
Wolkow, Driscoll, & Hall, 2017). Here, we summarize the recent
findings regarding potential biomarkers of aging in C. elegans. We
classify and describe age‐related changes in C. elegans at the tissue,
cellular, and molecular levels (Figure 1). We also review the roles of
insulin/IGF‐1 signaling (IIS) and dietary restriction (DR), two evolu-
tionarily conserved aging‐modulating regimes, in age‐related changes.
We then discuss the potential utility of these changes as biomarkers
of aging in C. elegans. We believe that these biomarkers will provide
valuable insights into the aging process and that this information will
advance our understanding of aging and age‐related diseases in
mammals, including humans.
2 | AGE‐DEPENDENT CHANGES
2.1 | Age‐dependent changes at the tissue level
2.1.1 | Reproductive system
Like many other physiological systems, the reproductive system of
C. elegans undergoes age‐dependent changes (Figure 2). The rate of
reproduction significantly decreases with age, and the structure of
the reproductive system deteriorates (Garigan et al., 2002; Hughes,
Evason, Xiong, & Kornfeld, 2007; Luo, Kleemann, Ashraf, Shaw, &

Muscles

Neurons
Reproductive system
Tissue level
Cellular level
Molecular level Reproductive system
Nucleus
Nervous system
Gene expression Mitochondria
Muscles
RNA quality Endoplasmic reticulum
Age pigments
Protein aggregation
Proteomics & Metabolomics

F I G U R E 1 Age‐related changes in Caenorhabditis elegans. A schematic diagram shows age‐related changes that are discussed in this review.
We classified age‐related changes into three levels: tissue, cellular, and molecular

Fragmentation
Number of myofibrils
Muscles Darkening

Neurons Reproductive system

Beading Rate of reproduction
Blebbing Structure
Branching Number of sperms
Number of vesicles Number of nuclei
Regeneration Size of oocytes
Learning ability Fertilized oocytes

F I G U R E 2 Age‐related changes at the tissue level. As Caenorhabditis elegans ages, the integrity of muscles, neurons, and the reproductive
system declines. Muscles become fragmented, and the tissues of the reproductive system degenerate. Neuronal integrity appears to be
prolonged compared to other tissues, but neurons also undergo age‐dependent deterioration, such as blebbing
SON ET AL.
Murphy, 2010; McGee, Day, Graham, & Melov, 2012; Pickett, Diet-
rich, Chen, Xiong, & Kornfeld, 2013). As C. elegans is a self‐fertile
hermaphrodite with a limited number of sperm (Ward & Carrel,
1979), sperm shortage is one of the reasons for reduced progeny at
an early age. This is because the number of progeny produced by
unmated (self‐fertilized) C. elegans decreases sharply and relatively
early (Day 3) in adulthood (Hughes et al., 2007). In contrast, age‐de-
pendent reductions in the number of progeny are delayed in mated
hermaphrodites that receive sperm from other males compared to
unmated hermaphrodites (Hughes et al., 2007; Luo et al., 2010).
However, mated hermaphrodites also cease reproduction after a cer-
tain time point, implying the existence of factors, other than sperm
shortage, that limit the reproductive period. Age‐associated deterio-
ration of tissues in the reproductive system may hamper prolonged
reproduction, despite the supply of extra sperm at an advanced age.
In aged worms, the number of nuclei in the mitotic germline dimin-
ishes and the nucleoplasm displays an increased accumulation of
grainy material and cavities (Garigan et al., 2002). Oocyte size also
decreases during aging, which correlates with their reduced quality,
suggesting that an appropriate oocyte size is crucial for reproduction
(Andux & Ellis, 2008). In addition, the number of unfertilized oocytes
in mated hermaphrodites increases with age (Luo et al., 2010). Thus,
the germline of old worms undergoes sperm shortages and structural
changes, which eventually render the worms sterile.
Mechanisms of reduced reproductive potency in aged male C. ele-
gans have also been proposed (Chatterjee et al., 2013; Guo, Navetta,
Gualberto, & Garcia, 2012). Although the activity, morphology, and
number of sperm are preserved during the first 3 days of adulthood,
3‐day‐old adults exhibit uncoordinated mating behaviors and a rapid
decline in reproductive potency (Chatterjee et al., 2013; Guo et al.,
2012). This uncoordinated mating behavior appears to be caused by
hyperexcited muscles in the male reproductive system (Guo et al.,
2012), as decreasing the activity of these hyperexcited muscles has
been shown to improve mating potency. Overall, the reproductive sys-
tems of both hermaphrodites and males display age‐related changes,
which lead to a reduction in the reproductive potential.
2.1.2 | Nervous system
Although it was initially reported that C. elegans neurons remain rela-
tively intact during aging compared with other tissues (Collins et al.,
2008; Herndon et al., 2002), subsequent studies have shown that neu-
rons display subtle but reproducible age‐dependent changes (Chen,
Chen, Jiang, Chen, & Pan, 2013; Chew, Fan, Gotz, & Nicholas, 2013;
Collins et al., 2008; Herndon et al., 2002; Pan, Peng, Chen, & McIntire,
2011; Tank, Rodgers, & Kenyon, 2011; Toth et al., 2012; Figure 2).
Touch receptor neurons, such as ALM neurons, display gradual bead-
ing, blebbing, and branching during aging (Pan et al., 2011; Tank et al.,
2011). In addition, the number of vesicles per synapse in touch recep-
tor neurons is significantly lower in aged worms than in young worms
(Toth et al., 2012). This indicates that the synaptic integrity of worms
also degenerates during aging. The extent of aberrant neuronal
changes varies among worms of the same age, and neuron integrity
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shows a positive correlation with the coordinated motility of worms.
In addition to the touch receptor neurons, GABAergic motor neurons
in aged worms display neurite branching (Tank et al., 2011). Another
study, however, suggested that GABAergic motor neurons undergo
minimal age‐related changes (Toth et al., 2012). Further studies are
needed to resolve these discrepancies, which perhaps originate from
differences in experimental conditions.
In addition to these structural changes, aged C. elegans display a
functional deterioration of neurons (Hammarlund, Nix, Hauth, Jor-
gensen, & Bastiani, 2009; Kauffman, Ashraf, Corces‐Zimmerman, Lan-
dis, & Murphy, 2010; Liu et al., 2013; Murakami & Murakami, 2005).
Caenorhabditis elegans motor neurons undergo functional declines
starting at an early age (Liu et al., 2013) and display reduced regenera-
tion capacity (Hammarlund et al., 2009; Kauffman et al., 2010; Liu
et al., 2013; Murakami & Murakami, 2005). Associative learning ability
also decreases with age (Hammarlund et al., 2009; Kauffman et al.,
2010; Murakami & Murakami, 2005). Thus, different neuron types
undergo different age‐dependent structural and functional declines.
2.1.3 | Muscles
An age‐dependent reduction in the muscle integrity of C. elegans has
been widely reported (Chow, Glenn, Johnston, Goldberg, & Wolkow,
2006; Garigan et al., 2002; Glenn et al., 2004; Herndon et al., 2002;
Johnston, Iser, Chow, Goldberg, & Wolkow, 2008; Shamir, Wolkow,
& Goldberg, 2009; Figure 2). Nuclei and sarcomeres in the body wall
muscle cells undergo age‐dependent changes. For example, nuclei in
the body wall muscle cells undergo redistribution with age. However,
the extent of this redistribution varies between as well as within
individual worms, suggesting that stochastic factors play a role in
this phenomenon (Herndon et al., 2002). Additionally, the size of
muscle cell nucleoli tends to increase during aging (Herndon et al.,
2002, see also Tiku et al., 2017). Furthermore, sarcomeres in the
body wall muscles lose their densely packed structures and regular
orientations with increasing age (Glenn et al., 2004; Herndon et al.,
2002). The C. elegans pharynx, a neuromuscular organ composed of
20 muscle cells and 20 neurons, also loses its integrity during aging
(Albertson & Thomson, 1976; Chow et al., 2006; Garigan et al.,
2002; Herndon et al., 2002; Johnston et al., 2008; Podshivalova,
Kerr, & Kenyon, 2017; Zhao et al., 2017). In aged worms, the num-
ber of myofibrils diminishes and the pharyngeal muscles exhibit an
abnormal appearance (Chow et al., 2006; Garigan et al., 2002; Hern-
don et al., 2002; Podshivalova et al., 2017; Zhao et al., 2017). Other
age‐dependent degenerative structural changes, such as darkening of
the pharyngeal regions, also occur (Chow et al., 2006; Garigan et al.,
2002; Herndon et al., 2002). These age‐dependent functional and
structural abnormalities of pharyngeal muscles appear to be associ-
ated with bacterial accumulation in the terminal bulb of old worms
(Podshivalova et al., 2017; Zhao et al., 2017). This is consistent with
the susceptibility of old worms to infection by pathogenic bacteria,
including Pseudomonas aeruginosa (Youngman, Rogers, & Kim, 2011).
Degenerative changes in the muscle morphology of C. elegans have
been confirmed via quantitative analytical methods (Johnston et al.,
4 of 11 | SON
Nucleus
Dark patches
Structural abnormality
Nuclei number
Size
Nucleolar size
F I G U R E 3 Age‐related changes at the cellular level. A schematic diagram that shows changes in the nuclei and mitochondria of old
Caenorhabditis elegans cells
2008; Shamir et al., 2009). Thus, the loss of muscular integrity in old
C. elegans results in age‐related changes, including impaired motility
and increased susceptibility to infection, as in humans.
2.2 | Age‐dependent changes at the cellular level
2.2.1 | Nucleus
The integrity of C. elegans nuclei generally diminishes with age,
although the degree of deterioration may vary among tissues
(Garigan et al., 2002; Golden et al., 2007; Haithcock et al., 2005;
Herndon et al., 2002; McGee et al., 2011; Figure 3). The intestine of
adult C. elegans has approximately 30–34 cells whose nuclei start to
degenerate at Day 12 of adulthood, resulting in a reduction in their
number and size (McGee et al., 2011). The nuclei of muscle cells also
develop dark patches with age (Haithcock et al., 2005; Herndon
et al., 2002), and the relative size of the nucleoli increases with aging
(Herndon et al., 2002; Tiku et al., 2017). Likewise, hypodermal and
pharyngeal cell nuclei exhibit age‐associated abnormalities (Haithcock
et al., 2005). The nuclear lamina, which supports the structure of
nucleus, displays irregular peripheral structures in the body wall mus-
cle, hypodermal, and intestinal cells (Haithcock et al., 2005). The
space between the nuclei of syncytial germ cells increases, and the
number of the nuclei decreases during aging (Garigan et al., 2002).
Overall, nuclei in C. elegans undergo age‐dependent structural
changes to varying degrees.
2.2.2 | Mitochondria
Mitochondria undergo age‐dependent structural and functional
changes as well (Gruber et al., 2011; Yasuda et al., 2006; Figure 3).
For example, aged mitochondria in the body wall muscle cells
become enlarged and swollen. These changes seem to be caused by
mitochondrial fusion rather than an increase in the size of individual
mitochondria because the total mitochondrial mass does not change
during aging (Yasuda et al., 2006). Old worms also display frag-
mented mitochondria (Hahm, Kim, DiLoreto, & Shi, 2015; Regmi,
Rolland, & Conradt, 2014; Roux, Langhans, Huynh, & Kenyon, 2016),
and this correlates with uncoordinated locomotion in aged C. elegans
(Hahm et al., 2015). Mitochondria are the major organelles that pro-
duce reactive oxygen species (ROS) that oxidize proteins and induce
ET AL.
Mitochondria
Size
Fragmentation
ROS production
Protein oxidation
Damaged mtDNA
mtDNA copy
Oxygen consumption rate
structural DNA damage, thus impairing the function of mitochondria
(Birben, Sahiner, Sackesen, Erzurum, & Kalayci, 2012). Increases in
the level of ROS, carbonylated proteins, and damaged mitochondrial
DNA have been reported during aging in C. elegans (Berlett & Stadt-
man, 1997; Cui, Kong, & Zhang, 2012; Gruber et al., 2011; Stadtman
& Berlett, 1997; Yasuda et al., 2006). Consistent with these changes,
mitochondrial DNA copy numbers (Gruber et al., 2011) and oxygen
consumption rates (Gruber et al., 2011; Yasuda et al., 2006) decrease
during aging. Thus, age‐dependent declines in the integrity of mito-
chondria appear to be associated with functional impairments.
2.2.3 | Endoplasmic reticulum
The endoplasmic reticulum (ER) is an organelle where proteins and
lipids are synthesized and modified and serves as a calcium reservoir.
The ER is also a site where the unfolded protein response (UPR) or
ERUPR occurs (Smith & Wilkinson, 2017). When misfolded proteins
accumulate under various stress conditions, the ERUPR is activated to
degrade the misfolded proteins through three key pathways includ-
ing the protein kinase RNA‐like ER kinase (PERK), the inositol‐requir-
ing enzyme‐1 (IRE‐1)/X‐box binding protein‐1 (XBP‐1), and the
activating transcription factor 6 (ATF6) pathways. The capacity of
the ERUPR seems to be reduced during aging, as has been deter-
mined by measuring the levels of HSP‐4, a target chaperone of IRE‐
1/XBP‐1, under ER stress conditions (Ben‐Zvi, Miller, & Morimoto,
2009; Maity et al., 2016; Martinez, Duran‐Aniotz, Cabral‐Miranda,
Vivar, & Hetz, 2017; Taylor & Dillin, 2013). In addition, the overex-
pression of XBP‐1 in neurons is sufficient for enhancing ER stress
resistance and longevity (Taylor & Dillin, 2013). Overall, these results
suggest that the ERUPR plays a functional role in aging and lifespan.
2.3 | Age‐dependent changes at the molecular level
2.3.1 | Gene expression
It is not surprising that the expression of many genes is altered dur-
ing C. elegans aging (Cortes‐Lopez et al., 2018; de Lencastre et al.,
2010; Golden, Hubbard, Dando, Herren, & Melov, 2008; Golden &
Melov, 2004; Kato, Chen, Inukai, Zhao, & Slack, 2011; Lund et al.,
2002; Rangaraju et al., 2015; Vinuela, Snoek, Riksen, & Kammenga,
2012; Youngman et al., 2011). For example, mRNA levels of heat‐
SON ET AL.
shock protein‐encoding genes increase until reaching midlife and
then decrease in old age (Golden & Melov, 2004; Golden et al.,
2008; Lund et al., 2002). In addition, the expression of many colla-
gen genes, whose overexpression increases lifespan, decreases with
age (Ewald, Landis, Porter Abate, Murphy, & Blackwell, 2015; Golden
et al., 2008). Many other aging‐associated gene expression changes
have been identified, but we still do not fully understand the effects
of global gene expression changes on aging. It will be important to
determine whether these gene expression changes have causative
roles in aging or are the consequences of aging.
The expression of many noncoding RNAs, including microRNAs
(miRNAs), Piwi‐interacting RNAs (piRNAs), transfer RNAs (tRNAs),
ribosomal RNAs (rRNAs), small nucleolar RNAs (snoRNAs), and cir-
cular RNAs (circRNAs), changes with age (Cortes‐Lopez et al., 2018;
de Lencastre et al., 2010; Ibanez‐Ventoso et al., 2006; Inukai &
Slack, 2013; Kato & Slack, 2013; Kato et al., 2011; Lucanic et al.,
2013). The overall levels of miRNAs tend to decrease during aging
(de Lencastre et al., 2010; Ibanez‐Ventoso et al., 2006), and this
appears to be caused by age‐dependent reductions in the mRNA
levels of dcr‐1/Dicer (Mori et al., 2012) and alg‐1/Argonaut (Inukai,
Pincus, de Lencastre, & Slack, 2018). The mRNA levels of alg‐1
appear to be decreased by miR‐71, whose levels increase with age
(de Lencastre et al., 2010; Inukai et al., 2018). Interestingly, miR‐71
is necessary and sufficient for longevity (de Lencastre et al., 2010),
suggesting a role for miR‐71 in aging through its regulation of alg‐1.
The overall levels of tRNAs, rRNAs, and snoRNAs increase with
age, whereas the levels of piRNAs decrease with age (Kato et al.,
2011). circRNAs are a recently identified type of noncoding RNAs,
whose ends are covalently linked and are implicated in transcrip-
tional regulation (Knupp & Miura, 2018). A recent study indicated
that the overall levels of circRNAs increase during C. elegans aging
(Cortes‐Lopez et al., 2018). Whether various noncoding RNAs, other
than miRNAs, act as biomarkers of aging and/or affect lifespan
remains to be determined.
2.3.2 | RNA quality
RNA quality control mechanisms decline during aging in C. elegans
(Heintz et al., 2017; Son et al., 2017). Nonsense‐mediated mRNA
decay (NMD) is a cellular protective pathway that degrades mRNAs
containing premature termination codons (PTCs), as these generate
potentially toxic truncated proteins (Miller & Pearce, 2014). NMD
activity decreases during aging in the tissues of various organs,
including the muscles, hypodermis, and intestine (Son et al., 2017). In
addition, mRNA splicing fidelity decreases with age, as shown by the
increased levels of introns and unannotated regions in the mRNAs
of aged worms (Heintz et al., 2017). Age‐dependent declines in RNA
quality control negatively affect longevity, because enhancing NMD
activity or overexpressing key splicing factors increases lifespan
(Heintz et al., 2017; Son et al., 2017).
mRNAs that are destined to be degraded, such as those tar-
geted by miRNAs, are stored and degraded in processing bodies
(P‐bodies; Chantarachot & Bailey‐Serres, 2018). In the P‐body,
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most mRNAs are degraded by decapping enzymes and exonucle-
ases. DCP1/decapping protein 1 and DCP2/decapping protein 2
remove the 5' cap of target mRNAs, and then the XRN1/5'‐3'
exoribonuclease fully degrades the mRNAs (Labno, Tomecki, &
Dziembowski, 2016). Interestingly, the inhibition of RNA decay by
RNAi targeting xrn‐1/XRN1 increases the number of dcp‐1::gfp
granules, which are also increased during aging (Rousakis et al.,
2014). These results suggest that RNA decay efficiency decreases
during aging. Furthermore, perturbation of the RNA decay compo-
nents affects lifespan; genetic inhibition of dcap‐1/DCP1, dcap‐2/
DCP2, or xrn‐1/XRN1 decreases lifespan in C. elegans (Rousakis
et al., 2014). In contrast to P‐body granules, aging does not affect
the number of stress granules that are induced by various stresses
(Chantarachot & Bailey‐Serres, 2018; Rousakis et al., 2014). Over-
all, the capacity for mRNA decay and proper mRNA metabolism
decreases with age.
2.3.3 | Age pigments
Organisms contain various types of fluorescent materials, including
aromatic amino acids, lipofuscin, advanced glycation end products,
and anthranilic acids, several of which accumulate with age. Lipo-
fuscin is well known as an “age pigment,” as its level increases
with aging in many organisms (Gray & Woulfe, 2005; Klass, 1977;
Xu, Chen, Manivannan, Lois, & Forrester, 2008). Age pigments can
be quantitated using in vivo fluorescence spectroscopy that
detects a broad range of excitation/emission spectra. The accumu-
lation of age pigments is gradual until early adulthood (Days 5–10)
and rapid in midlife (Days 10–15; Gerstbrein, Stamatas, Kollias, &
Driscoll, 2005). Furthermore, the absolute amount of generated
age pigments negatively correlates with health and lifespan, and
possibly predicts the lifespan of C. elegans (Gerstbrein et al., 2005;
Pincus, Smith‐Vikos, & Slack, 2011). However, a study by the
Gems group challenges the view of lipofuscin as an age pigment
in C. elegans (Coburn et al., 2013). They have shown that
kynurenines, which emit blue fluorescence, exhibit characteristics
similar to those of lipofuscin. Kynurenine levels do not increase
gradually with aging, but they are precipitously elevated upon the
death of the organism, resulting in sudden bursts of blue fluores-
cence (Coburn et al., 2013). A recent study proposes that sub-
stances emitting red signals increase gradually with age (Pincus,
Mazer, & Slack, 2016), whereas those emitting blue signals
increase only marginally during aging and dramatically after death
(Coburn et al., 2013; Pincus et al., 2016). Further characterization
of the chemical nature of age pigments is required to resolve this
issue.
2.3.4 | Protein aggregation
Protein homeostasis generally declines during aging, and this decline
is associated with age‐related diseases. For example, aggregated pro-
teins contribute to the pathology of neurodegenerative diseases,
such as Alzheimer's, Parkinson's, and polyglutamine diseases in
6 of 11 | SON
humans (Nollen et al., 2004; Taylor, Hardy, & Fischbeck, 2002). C. el-
egans displays an aging‐associated aggregation of transgene‐encoded
polyQ and α‐synuclein, which are models for Huntington's and
Parkinson's diseases, respectively (Morley, Brignull, Weyers, & Mori-
moto, 2002; van Ham et al., 2008). Many inherent proteins become
insoluble and form aggregates in aged worms (David et al., 2010;
Reis‐Rodrigues et al., 2012; Walther et al., 2015). Genetically inhibit-
ing each of many aggregation‐prone protein‐coding genes increases
lifespan (Reis‐Rodrigues et al., 2012), suggesting that protein aggre-
gation has a negative effect on longevity. Thus, aging processes are
accompanied by an increase in the accumulation of nonfunctional
and insoluble protein aggregates.
2.3.5 | Proteomic and metabolomic changes
“Omics” techniques allow researchers to examine universal changes
in genes, RNAs, proteins, and metabolites during aging. Using stable
isotope labeling with amino acids in cell culture (SILAC) followed by
LC‐MS/MS methods, C. elegans proteins whose levels increase or
decrease with age have been reported (Narayan et al., 2016). During
aging, proteins that likely function in nucleosome assembly, ER‐nu-
clear signaling, and the response to unfolded proteins increase,
whereas the abundance of proteins involved in metabolism such as
fatty acid, carbohydrate, and amino acid metabolism decreases
(Narayan et al., 2016). A similar observation has been reported
showing that overall levels of fatty acid metabolic proteins decrease
(Copes et al., 2015). Because altering fat metabolism has huge
impacts on aging and lifespan (Lee et al., 2015; Lemieux & Ashrafi,
2016), the proper maintenance of fat metabolism in old age will be
beneficial for longevity.
Levels of metabolites such as amino acids change with age as
well. A study using ultra performance liquid chromatography (UPLC)‐
MS/MS indicated that the levels of all of the amino acids except gly-
cine and aspartic acid increase at an early age (approximately 3 days
of adulthood) and then decrease sharply afterward (Gao et al.,
2017). Another study using proton NMR spectroscopy reported that
the levels of three amino acids (glycine, serine, and tyrosine) increase
during aging, whereas the levels of four amino acids (alanine, aspara-
gine, glutamate, and glutamine) decrease during aging (Davies,
Bundy, & Leroi, 2015). Importantly, some of these age‐dependent
changes reflect the biological age of worms (Davies et al., 2015), and
treatment with any amino acids except phenylalanine and aspartate
increases lifespan (Edwards et al., 2015). Further studies are required
to comprehensively understand the roles of age‐dependent changes
in amino acid levels in aging and longevity.
3 | THE ROLE OF INSULIN/IGF‐1
SIGNALING AND DIETARY RESTRICTION IN
AGE‐ASSOCIATED CHANGES IN C. ELEGANS
Many intrinsic and extrinsic lifespan regulatory factors, including
insulin/IGF‐1 signaling (IIS), target of rapamycin (TOR), mitochondrial
respiration, reproduction, and dietary restriction (DR), have been
ET AL.
identified using C. elegans. Among them, here we review age‐related
changes under reduced IIS and DR conditions, which are two repre-
sentative regimens for extending lifespan and delaying aging. The IIS
pathway is an evolutionarily conserved aging regulatory pathway
(Altintas, Park, & Lee, 2016; Kenyon, 2010). Mutations in the daf‐2/
insulin/IGF‐1 receptor double the lifespan of C. elegans by upregulat-
ing various transcription factors, including DAF‐16/FOXO, heat‐
shock factor 1 (HSF‐1), and SKN‐1/NRF. DR also promotes longevity
across phyla (Kapahi, Kaeberlein, & Hansen, 2017). Caenorhabditis
elegans eat‐2 mutants, which display reduced feeding rates and food
intake, are a widely used DR model (Lakowski & Hekimi, 1998; Rai-
zen, Lee, & Avery, 1995). Reduced IIS and DR delay various age‐re-
lated degenerative changes. The motility of worms, as measured by
maximum velocity, is prolonged in daf‐2 and eat‐2 mutants com-
pared to wild‐type animals (Hahm et al., 2015; Huang, Xiong, &
Kornfeld, 2004), although daf‐2 mutants display reduced sponta-
neous movements for a relatively long time (Bansal, Zhu, Yen, & Tis-
senbaum, 2015). Additionally, daf‐2 mutants are resistant to
bacterial colonization (Podshivalova et al., 2017), one of the main
causes of early death in C. elegans (Garigan et al., 2002; Zhao et al.,
2017). Moreover, the reproductive span of daf‐2 and eat‐2 mutant
hermaphrodites (Hughes et al., 2007; Luo et al., 2010; Luo, Shaw,
Ashraf, & Murphy, 2009) is longer than that of wild‐type worms,
although the total number of progeny produced by daf‐2 mutants is
reduced (Hughes et al., 2007). Mutations in daf‐2 also prolong the
reproductive period of males (Chatterjee et al., 2013). Furthermore,
daf‐2 mutants exhibit reduced uterine mass (McGee et al., 2012)
and delayed deterioration of germ cells (Garigan et al., 2002) com-
pared to wild‐type worms. Age‐dependent increases in neuronal
abnormalities, including neurite branching and irregularly shaped
soma in mechanosensory neurons, are delayed in daf‐2 mutants
compared to the wild type (Pan et al., 2011; Tank et al., 2011). Axon
regeneration capacity is also prolonged in daf‐2 mutants compared
to the wild‐type animals (Byrne et al., 2014). In contrast, neither
neurite branching nor axon regeneration appears to be delayed by
mutations in eat‐2 (Byrne et al., 2014; Tank et al., 2011). Neuronal
functions, such as associative learning ability, are maintained longer
in both daf‐2 and eat‐2 mutants than in wild‐type worms (Kauffman
et al., 2010).
Nuclear integrity in diverse tissues is maintained for a longer
period of time in daf‐2 mutants compared to in wild‐type worms, as
exemplified by the delayed degeneration of daf‐2 mutant nuclear
lamina (Haithcock et al., 2005). Mutations in age‐1, the gene that
encodes the phosphoinositide 3‐kinase acting immediately down-
stream of DAF‐2, delay nuclear degeneration in muscle (Herndon
et al., 2002). In addition, the age‐related loss of intestinal nuclei
(McGee et al., 2011) and hypodermal nuclei (Golden et al., 2007) is
reduced in daf‐2 mutants. Nucleolar size is negatively correlated
with longevity and is reduced in daf‐2 and eat‐2 mutants (Tiku et al.,
2017). In addition, daf‐2 mutants display higher ATP levels and
antioxidant activity and lower ROS levels than wild‐type worms
(Zarse et al., 2012). This indicates that mitochondrial activity is
increased, while ROS levels are reduced, likely because of an
SON ET AL. | 7 of 11

upregulation of antioxidants in daf‐2 mutants (Zarse et al., 2012). compared with slow‐moving worms. The rate of pharyngeal pump-
RNAi knockdown of daf‐2 during adulthood, however, transiently ing and the number of progeny after mating also positively corre-
increases the level of ROS, which acts as a signal for longevity late with remaining lifespan (Huang et al., 2004; Pickett et al.,
(Zarse et al., 2012), suggesting a stage‐specific regulation of ROS 2013). Second, nucleolar size has a negative correlation with long-
levels by IIS. evity (Tiku et al., 2017). Importantly, nucleolar size is reduced by
Mutations in daf‐2 delay the age‐associated accumulation of interventions that extend lifespan in Drosophila melanogaster, mice,
abnormal macromolecules, including mRNAs with PTCs (Son et al., and possibly humans as well (Tiku et al., 2017). Third, molecular
2017) and insoluble proteins (David et al., 2010). Age pigment accu- genetic changes are used as biomarkers of aging. The expression
mulation is reduced both in daf‐2 mutants and in eat‐2 mutants levels of several genes, including a small heat‐shock protein (hsp‐
(Gerstbrein et al., 2005). Overall, the delay in age‐related degenera- 16.2) after transient heat shock (Rea, Wu, Cypser, Vaupel, & John-
tive changes caused by daf‐2 mutations and DR is consistent with son, 2005) and superoxide dismutase 3 (sod‐3; Sanchez‐Blanco &
the idea that reduced IIS and DR confer healthy aging with a long Kim, 2011), are positively correlated with a long lifespan. In addi-
lifespan. tion, the expression levels of certain miRNAs increase or decrease
as worms grow old. Changes in these miRNA levels successfully
predict the remaining lifespan of C. elegans (Pincus et al., 2011).
4 | POTENTIAL BIOMARKERS OF AGING
Fourth, the amount of autofluorescent age pigments has been used
Biomarkers of aging reflect the physiological and functional age of as biomarkers of aging. These age pigments accumulate in old
organisms (Baker & Sprott, 1988). An important way to validate worms, and worms with a high accumulation of age pigments early
biomarkers of aging is by testing whether they predict the remain- in adulthood tend to live short lives (Pincus et al., 2016, 2011 ).
ing lifespan of an organism (Baker & Sprott, 1988; Johnson, 2006). Finally, mitochondrial functional integrity also predicts lifespan.
Several biomarkers predictive of lifespan have been reported in Superoxide bursts (Cheng et al., 2014; Shen et al., 2014) or pH
C. elegans (Table 1). First, physiological functions are indicators of changes (Schwarzlander et al., 2014) in the mitochondria, referred
the biological age of worms. For example, worms that display fast to as “mitoflashes,” early in adulthood (Day 3) show a negative cor-
locomotion during early adulthood (Hahm et al., 2015; Huang et al., relation with lifespan. However, morphological changes of the mito-
2004; Pincus et al., 2011) or maintain their youth speed in middle chondria do not correlate with lifespan (Regmi et al., 2014) and
age (Hsu, Feng, Hsieh, & Xu, 2009) tend to have longer lifespans therefore do not seem to be suitable as a biomarker of aging.
Overall, physiological and molecular factors, age pigments, and
mitochondrial integrity parameters are important biomarkers of
T A B L E 1 List of potential biomarkers of aging in Caenorhabditis aging in C. elegans. Whether these biomarkers are applicable to
elegans
other organisms needs to be validated. After validation, these uni-
Correlation versal biomarkers of aging will be very helpful in understanding the
Biomarkers of with
mechanistic and physiological causes and consequences of the
aging lifespan References
aging process in multiple species, including mammals.
Physiological Locomotion Positive Hahm et al. (2015),
markers Hsu et al. (2009),
Huang et al. (2004)
5 | CONCLUSIONS
and Pincus et al.
(2011) Risks for many chronic diseases, including cancer, cardiovascular dis-
Pharyngeal Positive Huang et al. (2004) eases, and neurodegenerative diseases, exponentially increase with
pumping rate
age. Research aiming at understanding the mechanisms of aging or
Progeny Positive Pickett et al. (2013) delaying aging can be beneficial for reducing these risks. The devel-
number
opment of biomarkers of aging is crucial for aging research, as they
Cellular Nucleolar size Negative Tiku et al. (2017)
reflect the biological age of an organism. In this review, we described
markers
age‐related changes that can potentially be used as biomarkers of
Molecular sod−3 Positive Sanchez‐Blanco and
markers expression Kim (2011) aging in C. elegans.
So far, diverse, promising biomarkers of aging have been iden-
hsp−16.2 Positive Rea et al. (2005)
expression tified in C. elegans. Several limitations, however, need to be
miRNAs Positive or Pincus et al. (2011) addressed in future research. First, the validity of several potential
negative biomarkers of aging is controversial, and, therefore, further studies
Age pigments Negative Pincus et al. (2011) resolving these discrepancies are needed. Second, a biomarker of
and Pincus et al. aging that reports one physiological or physical state usually does
(2016) not reflect the general biological age of an organism. For example,
Mitochondrial Negative Cheng et al. (2014) and a single worm that displays increased overall motility may have a
activity Shen et al. (2014)
reduced feeding rate. Therefore, it will be important to use
8 of 11 | SON
multiple biomarkers of aging collectively, from physiological mark-
ers to molecular markers, to precisely estimate the biological age
of an organism. In this sense, the standardization of which combi-
nations of biomarkers are the most effective for predicting the
biological age of worms will be helpful for the C. elegans aging
research field.
Recent studies have also demonstrated changes in the levels of
proteins and metabolites as well as RNAs during aging. As aging is a
stochastic process that influences many different biological compo-
nents, the analysis of these factors globally using omics technologies
will be crucial for the progress of the aging research field. Moreover,
the integration of omics data with physiological and molecular mark-
ers of aging will provide even more powerful tools toward a better
understanding of the mysteries of organismal aging.
ACKNOWLEDGMENTS
We thank all Lee laboratory members for their help and discussion.
This work was supported by the Korean Government (MSIP) through
the National Research Foundation of Korea (NRF; NRF‐
2016R1E1A1A01941152) to S‐J. V. L.
CONFLICT OF INTEREST
None declared.
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How to cite this article: Son HG, Altintas O, Kim EJE, Kwon
S, Lee S‐JV. Age‐dependent changes and biomarkers of aging
in Caenorhabditis elegans. Aging Cell. 2018;e12853.
https://doi.org/10.1111/acel.12853